Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Oct;174(19):6294–6297. doi: 10.1128/jb.174.19.6294-6297.1992

Decreased function of the class B tetracycline efflux protein Tet with mutations at aspartate 15, a putative intramembrane residue.

L M McMurry 1, M Stephan 1, S B Levy 1
PMCID: PMC207700  PMID: 1328154

Abstract

The aspartate 15 residue within the first predicted intramembrane helix of the tetracycline efflux protein Tet has been conserved in four tetracycline resistance determinants from gram-negative bacteria. Its replacement in class B Tet by tyrosine, histidine, or asparagine resulted in a 60 to 85% loss of tetracycline resistance and a similar loss of tetracycline-proton antiport. The tyrosine and histidine substitutions lowered the Vmax of the efflux system by some 90% but did not alter the Km. The asparagine substitution raised the Km over 13-fold, while the Vmax was equal to or greater than that of the wild type. Therefore, although the nature of its role is unclear, aspartate 15 is important for normal Tet function.

Full text

PDF
6294

Images in this article

6295Image
on p.6295
6296Image
on p.6296

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Curiale M. S., Levy S. B. Two complementation groups mediate tetracycline resistance determined by Tn10. J Bacteriol. 1982 Jul;151(1):209–215. doi: 10.1128/jb.151.1.209-215.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Curiale M. S., McMurry L. M., Levy S. B. Intracistronic complementation of the tetracycline resistance membrane protein of Tn10. J Bacteriol. 1984 Jan;157(1):211–217. doi: 10.1128/jb.157.1.211-217.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eckert B., Beck C. F. Topology of the transposon Tn10-encoded tetracycline resistance protein within the inner membrane of Escherichia coli. J Biol Chem. 1989 Jul 15;264(20):11663–11670. [PubMed] [Google Scholar]
  6. Hickman R. K., Levy S. B. Evidence that TET protein functions as a multimer in the inner membrane of Escherichia coli. J Bacteriol. 1988 Apr;170(4):1715–1720. doi: 10.1128/jb.170.4.1715-1720.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Lee H. C., Forte J. G. A study of H+ transport in gastric microsomal vesicles using fluorescent probes. Biochim Biophys Acta. 1978 Apr 4;508(2):339–356. doi: 10.1016/0005-2736(78)90336-x. [DOI] [PubMed] [Google Scholar]
  9. McMurry L., Petrucci R. E., Jr, Levy S. B. Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3974–3977. doi: 10.1073/pnas.77.7.3974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Noel D., Nikaido K., Ames G. F. A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry. 1979 Sep 18;18(19):4159–4165. doi: 10.1021/bi00586a017. [DOI] [PubMed] [Google Scholar]
  11. Rubin R. A., Levy S. B., Heinrikson R. L., Kézdy F. J. Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins. Gene. 1990 Mar 1;87(1):7–13. doi: 10.1016/0378-1119(90)90489-e. [DOI] [PubMed] [Google Scholar]
  12. Takahashi N., Takahashi Y., Blumberg B. S., Putnam F. W. Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4413–4417. doi: 10.1073/pnas.84.13.4413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Waters S. H., Rogowsky P., Grinsted J., Altenbuchner J., Schmitt R. The tetracycline resistance determinants of RP1 and Tn1721: nucleotide sequence analysis. Nucleic Acids Res. 1983 Sep 10;11(17):6089–6105. doi: 10.1093/nar/11.17.6089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yamaguchi A., Adachi K., Akasaka T., Ono N., Sawai T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon Tn10. Histidine 257 plays an essential role in H+ translocation. J Biol Chem. 1991 Apr 5;266(10):6045–6051. [PubMed] [Google Scholar]
  15. Yamaguchi A., Adachi K., Sawai T. Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli. FEBS Lett. 1990 Jun 4;265(1-2):17–19. doi: 10.1016/0014-5793(90)80872-g. [DOI] [PubMed] [Google Scholar]
  16. Yamaguchi A., Iwasaki-Ohba Y., Ono N., Kaneko-Ohdera M., Sawai T. Stoichiometry of metal-tetracycline/H+ antiport mediated by transposon Tn10-encoded tetracycline resistance protein in Escherichia coli. FEBS Lett. 1991 May 6;282(2):415–418. doi: 10.1016/0014-5793(91)80527-a. [DOI] [PubMed] [Google Scholar]
  17. Yamaguchi A., Udagawa T., Sawai T. Transport of divalent cations with tetracycline as mediated by the transposon Tn10-encoded tetracycline resistance protein. J Biol Chem. 1990 Mar 25;265(9):4809–4813. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES